Human TBK1 deficiency leads to autoinflammation driven by TNF-induced cell death.

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.

Non-canonical IKKs side with N4BP1 against the family.

The ubiquitin-binding endoribonuclease N4BP1 is a critical immunosuppressor, but the mechanism by which it acts to constrain TLR-induced inflammatory cytokine production has remained unclear. In this issue of Immunity, Gitlin et al. find that N4BP1 works in concert with the non-canonical IκB kinase (IKK) to limit activity of the IKK complex.

N4BP1 coordinates ubiquitin-dependent crosstalk within the IκB kinase family to limit Toll-like receptor signaling and inflammation.

The ubiquitin-binding endoribonuclease N4BP1 potently suppresses cytokine production by Toll-like receptors (TLRs) that signal through the adaptor MyD88 but is inactivated via caspase-8-mediated cleavage downstream of death receptors, TLR3, or TLR4. Here, we examined the mechanism whereby N4BP1 limits inflammatory responses. In macrophages, deletion of N4BP1 prolonged activation of inflammatory gene transcription at late time points after TRIF-independent TLR activation. Optimal suppression of inflammatory cytokines by N4BP1 depended on its ability to bind polyubiquitin chains, as macrophages and mice-bearing inactivating mutations in a ubiquitin-binding motif in N4BP1 displayed increased TLR-induced cytokine production. Deletion of the noncanonical IκB kinases (ncIKKs), Tbk1 and Ikke, or their adaptor Tank phenocopied N4bp1 deficiency and enhanced macrophage responses to TLR1/2, TLR7, or TLR9 stimulation. Mechanistically, N4BP1 acted in concert with the ncIKKs to limit the duration of canonical IκB kinase (IKKα/ß) signaling. Thus, N4BP1 and the ncIKKs serve as an important checkpoint against over-exuberant innate immune responses.

TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during Development and in Aging.

Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1-/- mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1+/- mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.

Inflammation Improves Glucose Homeostasis through IKKß-XBP1s Interaction.

It is widely believed that inflammation associated with obesity has an important role in the development of type 2 diabetes. IκB kinase beta (IKKß) is a crucial kinase that responds to inflammatory stimuli such as tumor necrosis factor α (TNF-α) by initiating a variety of intracellular signaling cascades and is considered to be a key element in the inflammation-mediated development of insulin resistance. We show here, contrary to expectation, that IKKß-mediated inflammation is a positive regulator of hepatic glucose homeostasis. IKKß phosphorylates the spliced form of X-Box Binding Protein 1 (XBP1s) and increases the activity of XBP1s. We have used three experimental approaches to enhance the IKKß activity in the liver of obese mice and observed increased XBP1s activity, reduced ER stress, and a significant improvement in insulin sensitivity and consequently in glucose homeostasis. Our results reveal a beneficial role of IKKß-mediated hepatic inflammation in glucose homeostasis.

Liquid phase separation of NEMO induced by polyubiquitin chains activates NF-κB.

The NF-κB essential modulator (NEMO) is a regulatory subunit of the IκB kinase (IKK) complex that phosphorylates the NF-κB inhibitors IκBs. NEMO mediates IKK activation by binding to polyubiquitin chains (polyUb). Here, we show that Lys63(K63)-linked or linear polyUb binding to NEMO robustly induced the formation of liquid-like droplets in which IKK was activated. This liquid phase separation of NEMO was driven by multivalent interactions between NEMO and polyUb. Both the NEMO ubiquitin-binding (NUB) domain and the zinc-finger (ZF) domain of NEMO mediated binding to polyUb and contributed to NEMO phase separation and IKK activation in cells. Moreover, NEMO mutations associated with human immunodeficiency impaired its phase separation. These results demonstrate that polyUb activates IKK and NF-κB signaling by promoting the phase separation of NEMO.

YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKɛ-mediated phosphorylation.

The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKɛ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.

Survival of Single Positive Thymocytes Depends upon Developmental Control of RIPK1 Kinase Signaling by the IKK Complex Independent of NF-κB.

NF-κB (nuclear factor κB) signaling is considered critical for single positive (SP) thymocyte development because loss of upstream activators of NF-κB, such as the IKK complex, arrests their development. We found that the compound ablation of RelA, cRel, and p50, required for canonical NF-κB transcription, had no impact upon thymocyte development. While IKK-deficient thymocytes were acutely sensitive to tumor necrosis factor (TNF)-induced cell death, Rel-deficient cells remained resistant, calling into question the importance of NF-κB as the IKK target required for thymocyte survival. Instead, we found that IKK controlled thymocyte survival by repressing cell-death-inducing activity of the serine/threonine kinase RIPK1. We observed that RIPK1 expression was induced during development of SP thymocytes and that IKK was required to prevent RIPK1-kinase-dependent death of SPs in vivo. Finally, we showed that IKK was required to protect Rel-deficient thymocytes from RIPK1-dependent cell death, underscoring the NF-κB-independent function of IKK during thymic development.

Inactivation of BAD by IKK inhibits TNFα-induced apoptosis independently of NF-κB activation.

The IκB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival, and tumorigenesis. The prosurvival function of IKK centers on activation of the transcription factor NF-κB, whose target gene products inhibit caspases and prevent prolonged JNK activation. Here, we report that inactivation of the BH3-only protein BAD by IKK independently of NF-κB activation suppresses TNFα-induced apoptosis. TNFα-treated Ikkß(-/-) mouse embryonic fibroblasts (MEFs) undergo apoptosis significantly faster than MEFs deficient in both RelA and cRel due to lack of inhibition of BAD by IKK. IKK phosphorylates BAD at serine-26 (Ser26) and primes it for inactivation. Elimination of Ser26 phosphorylation promotes BAD proapoptotic activity, thereby accelerating TNFα-induced apoptosis in cultured cells and increasing mortality in animals. Our results reveal that IKK inhibits TNFα-induced apoptosis through two distinct but cooperative mechanisms: activation of the survival factor NF-κB and inactivation of the proapoptotic BH3-only BAD protein.

Got PIDD1? Natural killer cells clear polyploid cells to ensure a balanced genome.

Removal of polyploid cells is essential to preventing cancer and restricting tumor growth. A new study published in The EMBO Journal shows assembly of the NEMO-PIDDosome on extra centrioles. Activation of this protein complex leads to NF-κB activation that, in turn, induces NK cell-mediated cell clearance.

IκB kinase-α coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy.

Activation of the IκB kinase (IKK) complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-κB has remained elusive. Here, analysis of IKK-dependent substrates in CRC cells after UV treatment revealed that phosphorylation of BRD4 by IKK-α is required for its chromatin-binding at target genes upon DNA damage. Moreover, IKK-α induces the NF-κB-dependent transcription of the cytokine LIF, leading to STAT3 activation, association with BRD4 and recruitment to specific target genes. IKK-α abrogation results in defective BRD4 and STAT3 functions and consequently irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK-α activity, BRD4, and the JAK/STAT pathway enhanced the therapeutic potential of 5-fluorouracil combined with irinotecan in CRC cells and is curative in a chemotherapy-resistant xenograft model. Finally, coordinated expression of LIF and IKK-α is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK-α, BRD4, and JAK/STAT signaling with clinical relevance.

Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma.

Ectopic lymphoid-like structures (ELSs) are often observed in cancer, yet their function is obscure. Although ELSs signify good prognosis in certain malignancies, we found that hepatic ELSs indicated poor prognosis for hepatocellular carcinoma (HCC). We studied an HCC mouse model that displayed abundant ELSs and found that they constituted immunopathological microniches wherein malignant hepatocyte progenitor cells appeared and thrived in a complex cellular and cytokine milieu until gaining self-sufficiency. The egress of progenitor cells and tumor formation were associated with the autocrine production of cytokines previously provided by the niche. ELSs developed via cooperation between the innate immune system and adaptive immune system, an event facilitated by activation of the transcription factor NF-κB and abolished by depletion of T cells. Such aberrant immunological foci might represent new targets for cancer therapy.

The Kinase IKKß Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKß and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKß and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKß to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.

α-Ketoglutarate-Activated NF-κB Signaling Promotes Compensatory Glucose Uptake and Brain Tumor Development.

The rapid proliferation of cancer cells and dysregulated vasculature within the tumor leads to limited nutrient accessibility. Cancer cells often rewire their metabolic pathways for adaption to nutrient stress, and the underlying mechanism remains largely unknown. Glutamate dehydrogenase 1 (GDH1) is a key enzyme in glutaminolysis that converts glutamate to α-ketoglutarate (α-KG). Here, we show that, under low glucose, GDH1 is phosphorylated at serine (S) 384 and interacts with RelA and IKKß. GDH1-produced α-KG directly binds to and activates IKKß and nuclear factor κB (NF-κB) signaling, which promotes glucose uptake and tumor cell survival by upregulating GLUT1, thereby accelerating gliomagenesis. In addition, GDH1 S384 phosphorylation correlates with the malignancy and prognosis of human glioblastoma. Our finding reveals a unique role of α-KG to directly regulate signal pathway, uncovers a distinct mechanism of metabolite-mediated NF-κB activation, and also establishes the critical role of α-KG-activated NF-κB in brain tumor development.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation.

The ligation of Toll-like receptors (TLRs) leads to rapid activation of dendritic cells (DCs). However, the metabolic requirements that support this process remain poorly defined. We found that DC glycolytic flux increased within minutes of exposure to TLR agonists and that this served an essential role in supporting the de novo synthesis of fatty acids for the expansion of the endoplasmic reticulum and Golgi required for the production and secretion of proteins that are integral to DC activation. Signaling via the kinases TBK1, IKKɛ and Akt was essential for the TLR-induced increase in glycolysis by promoting the association of the glycolytic enzyme HK-II with mitochondria. In summary, we identified the rapid induction of glycolysis as an integral component of TLR signaling that is essential for the anabolic demands of the activation and function of DCs.

Induction of MTHFD2 in Macrophages Inhibits Reactive Oxygen Species-mediated NF-κB Activation and Protects against Inflammatory Responses.

The one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is critical for cancer cell proliferation and immune cell phenotypes, but whether it can contribute to macrophage inflammatory responses remains unclear. In this study, we show that MTHFD2 was upregulated by LPS in murine macrophages upon activation of the TLR4-MyD88-IKKα/ß-NF-κB signaling pathway. MTHFD2 significantly attenuated LPS-induced macrophage proinflammatory cytokine production through its enzymatic activity. Notably, ablation of myeloid MTHFD2 rendered mice more sensitive to septic shock and CCl4-induced acute hepatitis. Mechanistically, MTHFD2 restrained IKKα/ß-NF-κB activation and macrophage inflammatory phenotype by scavenging reactive oxygen species through the generation of NADPH. Our study reveals MTHFD2 as a "self-control" mechanism in macrophage-mediated inflammatory responses.

NEMO and RIP1 control cell fate in response to extensive DNA damage via TNF-α feedforward signaling.

Upon DNA damage, ataxia telangiectasia mutated (ATM) kinase triggers multiple events to promote cell survival and facilitate repair. If damage is excessive, ATM stimulates cytokine secretion to alert neighboring cells and apoptosis to eliminate the afflicted cell. ATM augments cell survival by activating nuclear factor (NF)-κB; however, how ATM induces cytokine production and apoptosis remains elusive. Here we uncover a p53-independent mechanism that transmits ATM-driven cytokine and caspase signals upon strong genotoxic damage. Extensive DNA lesions stimulated two sequential NF-κB activation phases, requiring ATM and NEMO/IKK-γ: The first phase induced TNF-α-TNFR1 feedforward signaling, promoting the second phase and driving RIP1 phosphorylation. In turn, RIP1 kinase triggered JNK3/MAPK10-dependent interleukin-8 secretion and FADD-mediated proapoptotic caspase-8 activation. Thus, in the context of excessive DNA damage, ATM employs NEMO and RIP1 kinase through autocrine TNF-α signaling to switch on cytokine production and caspase activation. These results shed light on cell-fate regulation by ATM.

Histone H3.3 phosphorylation amplifies stimulation-induced transcription.

Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1-3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4-7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in 'canonical' H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and 'ejects' the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators.

Ubiquitin-Mediated Regulation of RIPK1 Kinase Activity Independent of IKK and MK2.

Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1's cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity.